Motile statocyst cilia transmit rather than directly transduce mechanical stimuli.

We have investigated the role of motile cilia in mechanotransduction by statocysts of the nudibranch mollusk Hermissenda crassicornis. Movement of the cilia that experience the weight of statoconia causes increased variance of voltage noise and membrane depolarization of the statocyst hair cell. Two complementary approaches were used to immobilize the cilia. Vanadate anion was iontophoretically injected into hair cells. This reversible inhibitor of vibratile form and to assume a more classic, pliable beat pattern. Voltage noise decreased as the cilia slowed and bent more extremely, nearly disappearing as motility was lost. When the intracellular vanadate concentration approached 10(-5) M, the cilia were arrested in an effective stroke against the cell membrane. The cell no longer depolarized upon gravitational or local mechanical stimulation. Rapid reversal of ciliary inhibition by norepinephrine or slow reversal with time restored both the voltage noise and depolarization response. Cilia were rendered rigid and upright by covalent cross-linkage of their membrane "sleeve" to the 9 + 2 axoneme, using the photoactivated, lipophilic, bifunctional agent 4,4'-dithiobisphenyl azide. In the initial stages of cross-linkage, the cilia remained vibratile but slowed and moved through wider excursions. Voltage noise decreased in frequency but increased in amplitude. When the cilia were fully arrested, voltage noise was minimized while the resting potential and membrane resistance remained essentially constant. Mechanical stimulation of the rigid cilia, normal to the cell membrane, elicited a generator potential of the same amplitude but of greater duration than before treatment. Because cilia that are partially arrested by vanadate undergo increased bending, although the hair cell shows decreased noise, neither the axoneme nor the ciliary membrane proper would appear to be sites of direct transduction. In cells with beating but stiffened cilia, however, the voltage noise becomes amplified, implying an increased efficiency of transduction. We suggest that active but rigid flexure of the axoneme is involved in amplification and continuous signal detection. The basal insertion area is the most likely transduction site, being the terminal leverage point through which force is applied to the plasma membrane via the flexing ciliary shaft.

Specific localization of scallop gill epithelial calmodulin in cilia.

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.

Studies of Environmental Risk Factors in Amyotrophic Lateral Sclerosis (ALS) and a Phase I Clinical Trial of L-Serine.

ß-N-Methylamino-L-alanine (BMAA) has been linked to Guam ALS/PDC and shown to produce neurodegeneration in vitro and in vivo (Drosophila, mice, rats, primates). BMAA misincorporation into neuroproteins produces protein misfolding and is inhibited by L-serine. Case-control studies in Northern New England indicate that living near to water-bodies with cyanobacterial blooms increases the risk of developing amyotrophic lateral sclerosis (ALS). The distribution of addresses of ALS cases in New Hampshire, Vermont, and Florida was compared to that of controls. Areas of statistically significantly increased numbers of ALS cases were examined for sources of environmental toxins. A phase I trial of oral L-serine was performed in 20 ALS patients (0.5 to 15 g twice daily). Safety and tolerability were assessed by comparing the rate of deterioration with 430 matched placebo controls. The distribution of residential addresses of ALS cases in New England and Florida revealed many areas where the age- and gender-adjusted frequency of ALS was greater than expected (P < 0.01). GIS studies of these "hot spots" in relation to sources of environmental pollutants, like cyanobacterial blooms, Superfund and Brownfield sites, and landfills, are ongoing. In the phase I trial of L-serine, two patients withdrew from because of gastrointestinal side effects. Three patients died during the study, which was about the expected number. The ALSFRS-R in the L-serine-treated patients showed a dose-related decrease in the rate of progression (34% reduction in slope, P = 0.044). The non-random distribution of addresses of ALS patients suggests that residential exposure to environmental pollutants may play an important role in the etiology of ALS. L-Serine in doses up to 15 g twice daily appears to be safe in patients with ALS. Exploratory studies of efficacy suggested that L-serine might slow disease progression. A phase II trial is planned.

Tumor necrosis factor-alpha induces changes in mitochondrial cellular distribution in motor neurons.

Motor neuron (MN) mitochondrial abnormalities and elevation in spinal fluid levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The mechanism of neuron death in ALS remains unclear, along with the contributions of mitochondrial dysfunction and inflammation in the process. Cell cultures enriched for MN derived from embryonic rat spinal cords were established and directly exposed in vitro to recombinant TNF-alpha for varying lengths of time. Although cytokine exposure for up to 4 days failed to induce MN death, mitochondrial changes were observed shortly after initiating treatment. Our results demonstrate that TNF-alpha induced mitochondrial redistribution toward the soma in MN. We postulate that inflammation may precede, and in fact cause, the mitochondrial changes observed in ALS tissue.

Polymyositis after ciguatera toxin exposure.

Biopsy-proved polymyositis subsequently developed in two patients who were severely poisoned by ciguatera fish toxin. Ciguatera toxin may have several mechanisms of action and may represent more than one toxin. The patients' clinical courses and the unlikelihood of coincidence of contracting both diseases suggested to us a causal relationship. Although we cannot prove this relationship, we suggest a mechanism by which the toxin predisposed the muscle to inflammation.

Alexia without agraphia associated with spleniogeniculate infarction.

Pure alexia, following an infarction in the distribution of the left posterior cerebral artery, is attributed to damage of the left occipital lobe and the splenium of the corpus callosum. We describe a case of pure alexia in a 57-year-old woman with infarction of the left lateral geniculate body and the splenium of the corpus callosum, a variation on this classic disconnection syndrome.

Identification and role of thiols in Toxoplasma gondii egress.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

Focal paraneoplastic limbic encephalitis presenting as orgasmic epilepsy.

PURPOSE: To report orgasmic epilepsy as a manifestation of paraneoplastic limbic encephalitis in a patient with small cell lung cancer. CASE REPORT: A 57 years-old woman presented with 2 month history of daily spells that consisted of a sudden pleasure provoking feeling described 'like an orgasm' lasting for 30 s to 1 min. She was a heavy smoker and had noted recent weight loss. Bronchial biopsy, following the finding of a right lung mass, confirmed the diagnosis of small cell lung cancer (SCLC). Spells subsided after starting carbamazepine. The lung cancer was treated with chemotherapy and chest radiation therapy resulting in a complete radiologic response. RESULTS: Brain magnetic resonance imaging (MRI) revealed left temporal lobe area of increased signal on T2 and FLAIR sequence. T1-weighted images after contrast administration demonstrated a circumscribed area of enhancement in the left anterior medial temporal lobe. Electroencephalogram (EEG) showed focal left mid-temporal sharp waves and intermittent slowing. Anti-Hu antibodies were detected in her serum supporting a diagnosis of paraneoplastic limbic encephalitis as the cause of her orgasmic epilepsy. The patient has been followed for 2 years after treatment without tumor recurrence or neurological deterioration. CONCLUSION: Orgasmic epilepsy is another mode of presentation of paraneoplastic limbic encephalitis leading to the diagnosis of an occult SCLC. EEG and MRI findings suggest that in this case the seizures originated from the left hemisphere. It is possible that early recognition and treatment of the SCLC will improve the prognosis of this neurologic entity.

Mechanical stimulation activates beating in calcium-arrested lateral cilia of Mytilus edulis gill.

Lateral cilia of Mytilus edulis gill arrest upon mechanical stimulation, the result of calcium influx. A mechanical stimulus that deflects these cilia toward the effective stroke, and is normally sufficient to cause transient arrest in beating lateral cilia or transient movement into the recovery stroke in quiescent cilia, initiates beating in Ca2+ ionophore-arrested cilia at 9-15 Hz, for periods as long as 30 s. This movement is restricted to the stimulated cilia and the beat pattern appears constrained in the first half of the beat cycle. Application of dopamine causes ciliary arrest in the presence (but not absence) of Ca2+ and mechanical stimulation will also activate such cilia to beat. In the presence of ATP, mechanical stimulation of detergent-permeabilized lateral cell models arrested in the presence of 50 microM Ca2+ will also cause activation comparable in frequency, duration, and beat pattern to that seen in Ca2+-arrested cells, but the initiation is more difficult. Upon application of ionophore in Ca2+-free (EGTA) seawater, the cilia become quiescent, stopped at the end of the recovery stroke. Mechanical stimulation will cause activation of beat, with a similar range of frequencies and duration as in Ca2+-arrested lateral cilia, but the beat pattern is normal and cilia of adjacent cells may also beat, presumably initiated by mechanical coupling. Gently lifting cilia at their basal ends, using small, slow movements of a mechanical probe, will initiate several beat cycles in quiescent lateral cilia but will cause Ca2+-arrested cilia to 'snap' into the effective stroke and back.(ABSTRACT TRUNCATED AT 250 WORDS)

EGTA induces prolonged summed depolarizations in Mytilus gill coupled ciliated epithelial cells: implications for the control of ciliary motility.

Abfrontal ciliated cells of Mytilus edulis gill beat when mechanically stimulated, a consequence of a Ca++-based generator potential and regenerative response. In contrast, the lateral ciliated epithelial cells arrest when stimulated, a consequence of a Ca++-based generator potential and a Na+/Ca++-based regenerative response. Iontophoretic injection of EGTA in abfrontal cells, followed by mechanical stimulation, results in a large, prolonged depolarization that returns to the resting level stepwise. It has been hypothesized that this phenomenon is caused by successive Ca++-dependent repolarizations in coupled cells, first in adjacent cells and then in the injected cell, in accord with relative EGTA loading. We have now demonstrated this same stepwise repolarization phenomenon in the Na+/Ca++-dependent lateral ciliated cells. In this case, each repolarization step is often preceded by a small spike. With either cell type, using two-electrode recording techniques, we can detect the stepwise repolarization in distant cells, proportionately decremented when the second (KCl) electrode is some distance from the injection (EGTA) electrode and stimulus. When force is applied between the electrodes and nearest the KCl electrode, a greater initial response is recorded from this electrode, presumably resulting from depolarization of its impaled cell, prolonged by EGTA diffusion through the intervening cell junctions. The subsequent repolarization steps are of approximately the same size, suggesting repolarization of cells between the two electrodes. These observations are consistent with the cell coupling/EGTA loading hypothesis and indicate that both cell types mediate repolarization through Ca++ and propagate ciliary beat or arrest through intracellular coupling.

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia.

Pure lateral (L) cilia may be separated from the remaining (R) cilia types of Mytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with 32P-labeled ATP at low Ca++ (10(-7) M), where L cilia beat, or in high Ca++ (2-20 microM), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5-15% gels, and autoradiographed. Neither cilia type undergoes Ca++-dependent phosphorylation of specific proteins, suggesting that neither Ca++-induced arrest in L cilia nor the Ca++ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca++-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca++-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP2). PIP2 increases at least 3-fold under Ca++-arrest conditions. Aequipecten gill lateral cilia, which require higher Ca++ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca++ levels. Phosphorylation of other lipids is Ca++-independent. In the Ca++-insensitive or activated R cilia, PIP2 levels are intermediate, increasing only marginally with increased [Ca++]. The formation of PIP2 in response to Ca++, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca++ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca++ ions, acting directly on the axoneme.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP and calcium in the differential control of Mytilus gill cilia.

Lateral (L) cilia of Mytilus gill are activated by serotonin which, in molluscan systems, is known to activate adenylate cyclase. Triton-extracted models of L-cells, arrested at greater than 10(-6) M Ca++, are stimulated to beat by the addition of 10(-5) M cAMP while still under Ca++ arrest conditions, suggesting that cAMP-activation is not mediated by alterations of Ca++ levels. Using isolated, permeabilized cilia, we find, independent of [Ca++], that cAMP-dependent protein phosphorylation in L-cilia occurs uniquely and reversibly on three low molecular weight polypeptides of 23,000, 18,000, and 14,000 daltons. Phosphorylation is maximal at cAMP concentrations above 0.5 microM. The phosphorylated chains partially co-extract at high salt with a 14S dynein fraction and have approximately the same molecular weights as reported for dynein light chains. Such conditions mainly extract the outer dynein arm, about 40% of the Mg++-ATPase activity, and a corresponding amount of the cAMP phosphorylated chains. However, the three polypeptides sediment together at 10-11S, clearly separable from the 14S dynein ATPase. Using a gel-overlay technique, we find that calmodulin binds to axonemal polypeptides of L-cilia with molecular weights of 18,000 and 13,000, independent of Ca++, while in mixed-population cilia, only a 12,000 dalton chain binds calmodulin, in a Ca++ dependent manner. In neither case are calmodulin binding proteins found in the high salt fraction containing the cAMP-dependent phosphorylated chains, indicating that, in spite of some similarity in molecular weight, the cAMP-phosphorylated and calmodulin binding polypeptides are different. Also, double-labelling indicates that only the 18,000 dalton chains co-migrate.(ABSTRACT TRUNCATED AT 250 WORDS)

MRI findings in a case of ophthalmoplegic migraine.

MRI using gadolinium contrast material can demonstrate lesions in cranial nerves. Tumors and inflammatory lesions have been described. There is little published information on MRI of cranial nerves in patients with migraine headaches with ophthalmoplegia. We present a case of ophthalmoplegic migraine with a cranial nerve abnormality which was subsequently shown to improve as the patient clinically improved. Implications from this finding are discussed in relation to the pathophysiology of ophthalmoplegic migraine.

Mechanical sensitivity and cell coupling in the ciliated epithelial cells of Mytilus edulis gill. An ultrastructural and developmental analysis.

Transmission electron microscopy has not provided strong evidence for gap junctions in Mytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.

Toxoplasma gondii: dithiol-induced Ca2+ flux causes egress of parasites from the parasitophorous vacuole.

Ca2+ is an essential activator of motility in the obligate intracellular parasite Toxoplasma gondii. Ca2+ ionophore A23187 and intracellular microinjection of Ca2+ initiate motility of parasites residing in parasitophorous vacuoles (PV). The source of Ca2+ and the mechanism by which it activates motility in vivo remain uncertain. Exposure of the parasites to dithiothreitol (DTT) can activate egress of previously nonmotile intravacuolar parasites within 60 sec. DTT is also known to activate both isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) produced by T. gondii. Using an adherent cell analysis system (ACAS) for Ca2+ imaging, a brief 15-50% increase in intra-PV fluorescence ratio was observed after exposure of infected fibroblasts to 5 mM DTT. Chelation of intracellular Ca2+ with BAPTA-AM and extracellular Ca2+ with EGTA blocked the DTT effect; however, this chelation did not prevent the activation of parasites nor the Ca2+ response to the Ca2+ ionophore ionomycin, suggesting that the Ca2+ that activates motility may reside near or within the parasite itself. This result demonstrates that an increase in Ca2+ within the vacuole precedes the onset of motility and the correlation of the DTT effect on motility and tachyzoite NTPase suggests that NTPase activation may be involved in the Ca2+ flux.

Cryptogenic epilepsy: an infectious etiology?

PURPOSE: Cryptogenic epilepsy, the group of epilepsy syndromes for which an etiology is unknown, comprises approximately 20% of all epilepsy syndromes. We selected patients in this subgroup of epilepsy and tested them for evidence of Toxoplasma gondii IgG antibodies by the enzyme-linked immunosorbent assay. T. gondii is found in up to 20% of the U.S. population forming dormant brain cysts in the latent bradyzoite form. We investigated the hypothesis that dormant T. gondii infection might be associated with cryptogenic epilepsy. METHODS: We selected patients with cryptogenic epilepsies and tested them for evidence of T. gondii IgG antibodies by the enzyme-linked immunosorbent assay. A control group was also tested for comparison. RESULTS: We have found a statistically-significant elevation of T. gondii antibodies among cryptogenic epilepsy patients as compared to controls [59% increase in optical density (OD), p = 0.013]. This association persisted after adjustment for subjects' gender and age in a multiple logistic regression model; however, it was no longer as statistically significant. CONCLUSIONS: Our results suggest that chronic T. gondii infection with brain cysts may be a cause of cryptogenic epilepsy.